基于miRNA-204通过mTOR通路调控海马神经元内质网自噬探讨加味柴胡疏肝汤对癫痫的疗效机制研究

With the effect of liver and phlegm extinguishing the Modified Chaihu Shugan Decoction clinical treatment of epilepsy effective, but the mechanism is unknown. Previous experiments confirmed that miRNA-204 regulates ATG7 and LC3-II expression, which is associated with autophagy of endoplasmic reticulum. It was suggested that the miRNA-204 was up-regulated by Chaihu Shugan Decoction, ATG7 and LC3-Ⅱ were expressed by mTOR pathway, the stress response of endoplasmic reticulum was activated, and the metabolic products of seizure were removed. The mice were treated with Chaihu Shugan Decoction to establish pilocarpine acute epilepsy mouse model. MicroRNA injection of miRNA-204 mimetic and inhibitor intervention in hippocampus was observed by immunohistochemistry and qRT-PCR The expressions of AKT, S6K and ATG7 and LC3-Ⅱ were detected by western blot. The apoptotic cells were detected by TUNEL method, and the gene chip and qRT-PCR were used to detect the expression of ATT7 and LC3-Ⅱ in mTOR pathway. MiRNA-204 / mTOR / endoplasmic reticulum autophagy pathway in the regulation of Chinese medicine after the intervention and signal transmission to explore its neurological protection mechanism. For the prevention and treatment of epilepsy to open up new ideas, and for the party to better use in clinical solid foundation.

具有疏肝化痰熄风功效的加味柴胡疏肝汤临床治疗癫痫有效，但机制不明。前期实验证实miRNA-204调控ATG7和LC3-Ⅱ表达与内质网自噬相关。推测加味柴胡疏肝汤上调miRNA-204，通过mTOR通路表达ATG7、LC3-Ⅱ，激活内质网应激反应，清除癫痫发作代谢产物，起神经保护作用。本项目拟经加味柴胡疏肝汤干预，建立匹罗卡品急性期癫痫小鼠模型，海马区微量注射miRNA-204模拟物和抑制物干预miRNA-204表达，采用免疫组化、qRT-PCR、western blot检测mTOR通路上下游靶点AKT、S6K和内质网自噬相关蛋白ATG7、LC3-Ⅱ表达，电镜检测内质网自噬，TUNEL法检测细胞凋亡，基因芯片和qRT-PCR研究miRNA-204/mTOR/内质网自噬通路在中药干预后的调控作用和信号传递规律，以探讨其神经保护机制。为癫痫的防治开拓新思路，并为该方更好的应用于临床夯实基础。

具有疏肝化痰熄风功效的加味柴胡疏肝汤临床治疗癫痫有效，但机制不明。前期实验证实miRNA-204调控ATG7和LC3-Ⅱ表达，与内质网自噬相关。课题组通过建立急性期癫痫小鼠模型，一方面应用PCR、Western blot、TUNEL法、基因芯片等技术研究加味柴胡疏肝汤干预后对miRNA-204/mTOR/内质网自噬通路的调控作用及其信号传递的规律，检测和观察癫痫小鼠 miRNA-204/mTOR/内质网自噬通路相关蛋白AKT、S6K、ATG7、LC3-Ⅱ在小鼠海马区表达的变化及神经细胞凋亡情况，采用电镜和光镜观察小鼠海马形态学改变，并通过透射电镜检测内质网自噬情况。另一方面利用miRNA-204的模拟物和抑制物对小鼠进行海马区立体定位微量注射干预，了解miRNA-204在海马组织内表达水平的变化对miRNA-204/mTOR/内质网自噬通路的影响。实验研究成果表明，经加味柴胡疏肝汤干预后，上调 miRNA-204，通过mTOR 通路介导 ATG7、LC3-Ⅱ的表达，激活内质网应激反应，清除癫痫发作的代谢产物，起到神经保护作用，达到治疗癫痫的目的。从分子生物学角度进一步明确加味柴胡疏肝汤治疗癫痫的疗效机制，为加味柴胡疏肝汤更好地应用于临床提供科学依据，深化了现有关于癫痫发病机制的理论研究，有利于提高对癫痫的诊断及治疗水平，发挥中医药治疗重大疾病的协同作用，扩大中医药治疗癫痫的社会影响力。